The division of chromosomes into euchromatic and heterochromatic regions is an enigmatic aspect of genome organization in multicellular eukaryotes. Heterochromatin is a major component of genomes as diverse as humans and fruit flies, and plays critical roles in chromosome inheritance and metabolism. In the absence of a detailed analysis of heterochromatin organization and sequence, our ability to investigate chromosome function, heterochromatic genes, and genome evolution remains limited. In the previous granting period, the Drosophila Heterochromatin Genome Project (DHGP) used genomic resources available in Drosophila melanogaster to generate basic information about the structure and sequence composition of the heterochromatin, specifically focusing on gene- and transposable element- rich regions that do not contain long arrays of highly-repeated satellite DNAs. We have assembled 12.5Mb of finished or nearly finished sequence and a BAC-based physical map that spans 16.5Mb of the heterochromatin, and linked sequences and clones to specific locations in the cytological maps. Annotation of 49Mb of sequence identified 700 gene models and other genetic elements, as well as orthologs in other Drosophilids, and demonstrated that >85% of the target sequences contains repetitive DNA. Here, we propose to extend the D. melanogaster heterochromatin sequence and physical maps to encompass most or all of the non-satellite regions, and to initiate studies of satellite regions. We will also to improve annotations and public data displays, examine evolution of heterochromatin through comparative analysis with other Drosophila species, and develop tools to facilitate heterochromatin assemblies and analysis. Achieving these aims will provide information and tools that will further our understanding of higher eukaryotic genome structure, and will lay the groundwork for more complete analysis of heterochromatin structure and function in Drosophila and other eukaryotes, including humans. [unreadable] [unreadable] [unreadable]